UDP-Glucuronosyl Transferase (UGT) is an enzyme that catalyzes the reaction of adding glucuronic acid to, for example, a drug, a foreign substance, or an endogenous substance such as bilirubin, steroid hormone, or bile acid. It is known as a drug-metabolizing enzyme. A plurality of isozymes that are classified into the UGT1 family and the UGT2 family have been reported as the UGT. Genetic polymorphisms exist in a gene (UGT1A1 gene) that codes UGT1A1 belonging to the UGT1 family among those isozymes. Generally, they are said to be involved in the incidence of side effects of irinotecan hydrochloride, an anticancer agent. Specifically, it has been reported that when a patient has a UGT1A1 gene polymorphism, the function of detoxifying irinotecan hydrolysate (SN-38) having high antitumor activity by using the UGT is deteriorated, which causes serious side effects such as a decrease in the number of white blood cells and diarrhea. Known examples of typical genetic polymorphisms involved in such side effects include UGT1A1*28, a polymorphism in a promoter region, as well as UGT1A1*6 and UGT1A1*27, polymorphisms in exon 1. Particularly, it has been reported that in addition to UGT1A1*28, the most important polymorphism, Asians including Japanese also have at least one of polymorphisms, UGT1A1*6 and UGT1A1*27, in combination therewith and thereby stronger side effects tend to be manifested. Furthermore, since UGT1A1 is involved in bilirubin conjugation formed through glucuronic acid in vivo, the polymorphisms thereof also cause constitutional jaundice such as Gilbert syndrome. Accordingly, the examination of a plurality of polymorphisms with respect to the UGT1A1 gene is very important to predict the degree and the onset of side effect to be caused by an anticancer agent.
On the other hand, the detection of a point mutation, a so-called single nucleotide polymorphism (SNP), is employed widely as a method of analyzing, at the gene level, for example, the causes of all types of diseases and the individual differences in disease liability (susceptibility to diseases) and in drug action. Examples of the common methods of detecting a point mutation include: (1) a direct sequencing method in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by a polymerase chain reaction (PCR) and all the gene sequences are analyzed, (2) a RFLP analysis in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by PCR, the amplification product thus obtained is cut with a restriction enzyme whose cleaving action differs depending on the presence or absence of the target mutation in the sequence to be detected and is then electrophoresed, and thereby typing is performed, and (3) the ASP-PCR method in which PCR is performed using a primer with a target mutation located at the 3′-end region and the mutation is judged depending on the presence or absence of amplification.
However, since these methods require, for example, purification of DNA extracted from a sample, electrophoresis, and a treatment with a restriction enzyme, they take time and cost. Furthermore, after PCR is performed, it is necessary to open the reaction container once. Accordingly, there is a possibility that the amplification product may contaminate the next reaction system and thereby the analysis accuracy may be deteriorated. Moreover, since it is difficult to automate, a large amount of samples cannot be analyzed. Further, the aforementioned ASP-PCR method (3) is less specific, which also is a problem.
Because of these problems, recently, a method of analyzing the melting temperature (Tm) of double-stranded nucleic acid formed of a probe and target nucleic acid is used as a method of detecting a point mutation. Since such a method is performed through, for example, Tm analysis or analysis of the melting curve of the double strand, it is referred to as melting curve analysis. This method is described below. That is, first, a probe complementary to a sequence to be detected containing a target point mutation is used to form a hybrid (double-stranded DNA) between the aforementioned probe and a target single-stranded DNA contained in a detection sample. Subsequently, this hybridization product is heat-treated, and dissociation (melting) of the hybrid accompanying the temperature rise is detected by a change in a signal such as absorbance. The Tm value then is determined based on the result of the detection and the presence or absence of any point mutation is judged accordingly. The higher the homology of the hybridization product, the higher the Tm value, and the lower the homology, the lower the Tm value. Therefore the Tm value (reference value for assessment) is determined beforehand with respect to the hybridization product between the sequence to be detected containing a point mutation and a probe complementary thereto, and then the Tm value (measured value) of the hybridization product between the target single-stranded DNA contained in the detection sample and the aforementioned probe is measured. When the measured value is comparable to the reference value, it is considered as matching, that is, it can be judged that a point mutation is present in the target DNA. On the other hand, when the measured value is lower than the reference value, it is considered as mismatching, that is, it can be judged that no point mutation is present in the target DNA. Furthermore, according to this method, it also is possible to automate gene analysis.
However, such a detection method using Tm analysis also has a problem in that a region including a site to be detected must be able to be amplified specifically and efficiently in PCR. Particularly, many isozymes are present in UGT and the sequences for coding them also are very similar to one another. Accordingly, there is a possibility that genes coding isozymes other than UGT1A1 also are amplified in PCR. Furthermore, when other isozyme-coding genes also have been amplified as described above, it may cause a decrease in the reliability of the analysis result in the analysis of a particular polymorphism (UGT1A1*28, UGT1A1*6, or UGT1A1*27) of the UGT1A1 gene (Nonpatent Document 1). Moreover, as described above, since the analysis of one sample is accompanied by a considerable amount of time and energy, it is not practical to analyze a large amount of samples, which also is a problem.    [Nonpatent Document 1] PMID: 11156391 Cancer Res. 2000 Dec. 15; 60(24): 6921-6.